The overall objective of this proposal is to determine the signaling networks mediating GTPase regulation of the ubiquitously expressed Na-H exchanger isoform, NHE1. Work during the previous funding period identified a new class of heterotrimeric GTPases, G-alpha12 and G-alpha13, that couple to the regulation of NHE1, and determined components of two signaling pathways mediating G-alpha13 activation of the exchanger. G-alpha13 activates NHE1 through Cdc42 and RhoA, members of the Rho family of monomeric GTPases that also function in regulating the actin cytoskeleton. The Cdc42-dependent activation of NHE1 by G-alpha13, but not the Rho- dependent activation, is mediated -by the MAP kinase MEKK1. During the previous funding period a novel gene was cloned that encodes a ubiquitously expressed NHE1-interacting phosphoprotein, NIP1. Overexpression of NIP1 inhibits NHE1 activity, and distinct GTPase pathways regulated by Ha-Ras and G-alpha13 signal through NIP1. The unifying hypothesis of this proposal is that GTPase-mediated signaling pathways act on multiple direct regulators of NHE1. This hypothesis will be tested by characterizing signaling networks regulating the exchanger, including the downstream targets of GTPases and the upstream regulators of NHE1. AIM 1 focuses on characterizing the role of recently identified RhoA effectors in G-alpha13 activation of NHE1 and determining the MAP kinase modules coupling Cdc42/MEKK1 to the exchanger. Nucleotide exchange factors mediating G- alpha13 activation of RhoA and Cdc42 will be identified and the signaling networks acting downstream of G-alpha13 will be tested as mediators of NHE1 activation by the beta-adrenergic receptor. AIM 2 investigates the NIP1- NHE1 association. Experiments are designed to determine how the phosphorylation state of NIP1, its regulation by GTPases, and its binding to NHE1 modify exchange activity. The only proteins known to bind and regulate NHE1 are calmodulin and NIP1, but structure-function analysis of NHE1 suggests a role for additional regulatory proteins. Thus in AIM 2 interaction cloning strategies will be used to identify proteins associating with NIP1 and with NHE1. Our long-term goal is to understand how changes in NHE1 affects cell proliferation, both normal and malignant-. The current study focuses on G-alpha13, NIP1, and NHE1, three ubiquitously expressed proteins that function in regulating cell growth.